Type 2 diabetes (T2D) results from the unfavorable intersection of impaired insulin sensitivity (insulin resistance, IR) and inadequate insulin secretion. While there has been phenomenal progress in identifying T2D associated genetic variants, there has been limited progress towards defining the specific causal variants and deciphering the molecular mechanisms between causal variation and biology. Most T2D variants appear to be acting through effects on pancreatic beta cell development or insulin production, processing or secretion. However, a growing number of these variants appear to relate to IR. These IR-associated variants are particularly important as IR is a powerful risk factor for T2D and cardiovascular disease with very limited therapeutic options. Understanding the mechanisms for these IR-related associations is now critical for progress in the field. We have assembled a unique group of investigators with complementary expertise both to accelerate the discovery of causal genes and variants that influence the risk for IR and to conduct functional studies to gain novel insight into the mechanisms by which these causal genes and variants contribute to disease. Our preliminary studies have prioritized IR related variants/loci based on orthogonal data including results from our unique GWAS of over 2,700 individuals with clamp based measures of insulin sensitivity as well as detailed physiologic characterization of IR variants. In this proposal, we wll employ methodology to identify causal variation and comprehensively investigate the mechanisms by which these variants modulate expression of causal genes in disease relevant cell types, i.e. adipose and skeletal muscle tissue. Studies in Aim 1 will determine regions of active chromatin in insulin sensitive tissues with histone modification ChIP-Seq and ATAC-Seq methods, investigate local interactions between the causal variant and causal gene with allele- specific expression, and identify causal long-range interactions in the context of whole genome architecture with ChIA-PET. In Aim 2, in vitro reporter gene studies will investigate the specific mechanisms for the causal variant and gene interactions as assayed by allele-specific transfection reporter gene studies and chromatin immunoprecipitation transcription factor binding assays. In Aim 3, the enhancer activity of transcriptional elements identified through these studies, and the relevance of causal variant modification of this enhancer activity, will be evaluated through genome editing in relevant cell lines, and in vivo utilizing transgenic mice with reporter constructs specifically docked into the mouse genome. Taken together, studies proposed will build on GWAS findings to identify causal variants and genes in IR associated loci that are fundamentally linked to insulin resistance, which will facilitate development of tools for risk prediction and early treatment of disease and potentially identify new therapeutic targets.